The invention is in the field of hepatitis virology. More specifically, this invention relates to recombinant proteins derived from an enterically transmitted strain of hepatitis E from Pakistan, SAR-55, and to diagnostic methods and vaccine applications which employ these proteins.
Epidemics of hepatitis E, an enterically transmitted non-A/non-B hepatitis, have been reported in Asia, Africa and Central America (Balayan, M. S. (1987), Soviet Medical Reviews, Section E, Virology Reviews, Zhdanov, O-V. M. (ed), Chur, Switzerland: Harwood Academic Publishers, vol. 2, 235-261; Purcell, R. G., et al. (1988) in Zuckerman, A. J. (ed), xe2x80x9cViral Hepatitis and Liver Diseasexe2x80x9d, New York: Alan R. Liss, 131-137; Bradley, D. W. (1990), British Medical Bulletin, 46:442-461; Ticehurst, J. R. (1991) in Hollinger, F. B., Lemon, S. M., Margolis, H. S. (eds): xe2x80x9cViral Hepatitis and Liver Diseasexe2x80x9d, Williams and Wilkins, Baltimore, 501-513). Cases of sporadic hepatitis, presumed to be hepatitis E, account for up to 90% of reported hepatitis in countries where hepatitis E virus (HEV) is endemic. The need for development of a serological test for the detection of anti-HEV antibodies in the sera of infected individuals is widely recognized in the field, but the very low concentration of HEV excreted from infected humans or animals made it impossible to use such HEV as the source of antigen for serological tests and although limited success was reported in propagation of HEV in cell culture (Huang, R. T. et al. (1992), J. Gen. Virol., 73:1143-1148), cell culture is currently too inefficient to produce the amounts of antigen required for serological tests.
Recently, major efforts worldwide to identify viral genomic sequences associated with hepatitis E have resulted in the cloning of the genomes of a limited number of strains of HEV (Tam, A. W. et al. (1991), Virology, 185:120-131; Tsarev, S. A. et al. (1992), Proc. Natl. Acad. Sci. USA, 89:559-563; Fry, K. E. et al. (1992), Virus Genes, 6:173-185). Analysis of the DNA sequences have led investigators to hypothesize that the HEV genome is organized into three open reading frames (ORFs) and to hypothesize that these ORFs encode intact HEV proteins.
A partial DNA sequence of the genome of an HEV strain from Burma (Myanmar) is disclosed in Reyes et al., 1990, Science, 247:1335-1339. Tam et al., 1991, and Reyes et al., PCT Patent Application WO91/15603 published Oct. 17, 1991 disclose the complete nucleotide sequence and a deduced amino acid sequence of the Burma strain of HEV. These authors hypothesized that three forward open reading frames (ORFS) are contained within the sequence of this strain.
Ichikawa et al., 1991, Microbiol. Immunol., 35:535-543, discloses the isolation of a series of clones of 240-320 nucleotides in length upon the screening of a xcexgt11 expression library with sera from HEV-infected cynomolgus monkeys. The recombinant protein expressed by one clone was expressed in E. coli. This fusion protein is encoded by the 3xe2x80x2 region of ORF-2 of the Myanmar strain of HEV.
The expression of additional proteins encoded within the 3xe2x80x2 region of ORF-2 of a Mexican strain of HEV and of a Burmese strain of HEV is described in Yarbough et al., 1991 J. Virology, 65:5790-5797. This article describes the isolation of two cDNA clones derived from HEV. These clones encode the proteins in the 3xe2x80x2 region of ORF-2. The clones were expressed in E. coli as fusion proteins.
Purdy et al., 1992, Archives of Virology, 123:335-349, and Favorov et al., 1992, J. of Medical Virolocy, 36:246-250, disclose the expression of a larger ORF-2 protein fragment from the Burma strain in E. coli. These references, as well as those previously discussed, only disclose the expression of a portion of the ORF-2 gene using bacterial expression systems. Successful expression of the full-length ORF-2 protein has not been disclosed until the present invention.
Comparison of the genome organization and morphological structure of HEV to that of other viruses revealed that HEV is most closely related to the caliciviruses. Of interest, the structural proteins of caliciviruses are encoded by the 3xe2x80x2 portion of their genome (Neil, J. D. et al. (1991) J. Virol., 65:5440-5447; and Carter, M. J. et al. (1992), J. Arch. Virol., 122:223-235) and although there is no direct evidence that the 3xe2x80x2 terminal part of the HEV genome also encodes the structural proteins, expression of certain small portions of the 3xe2x80x2 genome region in bacterial cells resulted in production of proteins reactive with anti-HEV sera in ELISA and Western blots (Yarborough, et al., (1991); Ichikawa et al. (1991); Favorov et al. (1992) and Dawson, G. J. et al. (1992) J. Virol. Meth; 38:175-186). However, the function of ORF-2 protein as a structural protein was not proven until the present invention.
The small proteins encoded by a portion of the ORF-2 gene have been used in immunoassay to detect antibodies to HEV in animal sera. The use of small bacterially expressed proteins as antigens in serological immunoassays has several potential drawbacks. First, the expression of these small proteins in bacterial cells often results in solubility problems and in non-specific cross-reactivity of patients"" sera with E. coli proteins when crude E. coli lysates are used as antigens in immunoassays (Purdy et al. (1992)). Second, the use of Western blots as a first-line serological test for anti-HEV antibodies in routine epidemiology is impractical due to time and cost constraints. An ELISA using small-peptides derived from the 3xe2x80x2-terminal part of the HEV genome resulted in the detection of only 41% positives from known HEV-infected patients. Third, it has been shown that for many viruses, including Picornaviridae which is the closest family to the Caliciviridae, important antigenic and immunogenic epitopes are highly conformational (Lemon, S. M. et al. (1991), in Hollinger, F. B., Lemon, S. M., Margolis, H. S. (eds): xe2x80x9cViral Hepatitis and Liver Diseasexe2x80x9d, Williams and Wilkins, Baltimore, 20-24). For this reason, it is believed that expression in a eukaryotic system of a complete ORF encoding an intact HEV gene would result in production of a protein which could form HEV-virus-like particles. Such a complete ORF protein would have an immunological structure closer to that of native capsid protein(s) than would the above-noted smaller proteins which represent only portions of the structural proteins of HEV. Therefore, these complete ORF proteins would likely serve as a more representative antigen and a more efficient immunogen than the currently-used smaller proteins.
The present invention relates to an isolated and substantially pure preparation of a human hepatitis E viral strain SAR-55.
The invention also relates to an isolated and substantially pure preparation of the genomic RNA of the human hepatitis E viral strain SAR-55.
The invention further relates to the cDNA of the human hepatitis E viral strain SAR-55.
It is an object of this invention to provide synthetic nucleic acid sequences capable of directing production of recombinant HEV proteins, as well as equivalent natural nucleic acid sequences. Such natural nucleic acid sequences may be isolated from a cDNA or genomic library from which the gene capable of directing synthesis of the HEV proteins may be identified and isolated. For purpose of this application, nucleic acid sequence refers to RNA, DNA, cDNA or any synthetic variant thereof which encodes for protein.
The invention further relates to a method for detection of the hepatitis E virus in biological samples based on selective amplification of hepatitis E gene fragments utilizing primers derived from the SAR-55 cDNA.
The invention also relates to the use of single-stranded antisense poly-or oligonucleotides derived from the SAR-55 cDNA to inhibit the expression of hepatitis E genes.
The invention also relates to isolated and substantially purified HEV proteins and variants thereof encoded by the HEV genome of SAR-55 or encoded by synthetic nucleic acid sequences and in particular to recombinant proteins encoded by at least one complete open reading frame of HEV.
The invention also relates to the method of preparing recombinant HEV proteins derived from an HEV genomic sequence by cloning the nucleic acid and inserting the cDNA into an expression vector and expressing the recombinant protein in a host cell.
The invention also relates to the use of the resultant recombinant HEV proteins as diagnostic agents and as vaccines.
The present invention also encompasses methods of detecting antibodies specific for hepatitis E virus in biological samples. Such methods are useful for diagnosis of infection and disease caused by HEV, and for monitoring the progression of such disease. Such methods are also useful for monitoring the efficacy of therapeutic agents during the course of treatment of HEV infection and disease in a mammal.
This invention also relates to pharmaceutical compositions for use in prevention or treatment of Hepatitis E in a mammal.